R ES E A RC H | R E PO R TS

have the same helper cell subset ratio as its SEX CHROMOSOME
predecessor population is consistent with each
effector cell in the population having the same
chance of becoming a memory cell. This model is
consistent with studies of CD8+ T cells indicating
Two genes substitute for the mouse
that memory cells arise from the effector cell
pool by a TCR-independent stochastic process Y chromosome for spermatogenesis
(14, 19–21).
Our finding that a clonal CD4+ memory T cell
population tended to produce an effector cell
and reproduction
population with the same subset composition Yasuhiro Yamauchi,1* Jonathan M. Riel,1* Victor A. Ruthig,1* Eglė A. Ortega,1
after recall suggests that a CXCR5– memory cell
Michael J. Mitchell,2 Monika A. Ward1†
produces CXCR5– effector cells and that a CXCR5+
memory cell produces CXCR5+ effector cells.
The mammalian Y chromosome is considered a symbol of maleness, as it encodes
Thus, although we (10) and others (15) have found
a gene driving male sex determination, Sry, as well as a battery of other genes
that bulk CXCR5+ memory cells can produce
important for male reproduction. We previously demonstrated in the mouse
CXCR5– and CXCR5+ effector cells, the present
that successful assisted reproduction can be achieved when the Y gene contribution
results fit the suggestion that both CXCR5+ and
is limited to only two genes, Sry and spermatogonial proliferation factor Eif2s3y.
CXCR5– cells are relatively lineage-committed
Here, we replaced Sry by transgenic activation of its downstream target Sox9, and Eif2s3y,
(15). An advantage of this process is that the
by transgenic overexpression of its X chromosome–encoded homolog Eif2s3x. The
helper cell subset diversity of the effector cell
resulting males with no Y chromosome genes produced haploid male gametes and sired

Downloaded from http://science.sciencemag.org/ on March 18, 2021

pool is carried into the memory cell pool and
offspring after assisted reproduction. Our findings support the existence of functional redundancy
retained thereafter.
between the Y chromosome genes and their homologs encoded on other chromosomes.

RE FE RENCES AND N OT ES
1. R. Ahmed, D. Gray, Science 272, 54–60 (1996).
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ACKN OW LEDG MEN TS
We thank S. Jameson and D. Masopust for critical discussions
and reading the manuscript and J. Walter for help with mouse
breeding and screening. The data presented in this manuscript
are tabulated in the main paper and in the supplementary
materials. Supported by NIH grants R01 AI039614 (M.K.J.),
R01 AI106791 (B.T.F.), and F32 AI107995 (N.J.T.).
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/351/6272/511/suppl/DC1
Materials and Methods
Figs. S1 and S2
References (22, 23)
17 July 2015; accepted 23 December 2015
10.1126/science.aad0483
M
any sexual characteristics are influenced
by sex chromosome constitution, with
mammalian females typically carrying XX
and males XY. We recently reported that
in the mouse, only two Y-chromosome
genes—testis-determinant Sry and spermatogonial
proliferation factor Eif2s3y—are needed for suc-
cessful assisted reproduction (1). Here, we asked
if these two genes could be replaced by trans-
genic activation of their homologs encoded on
other chromosomes.
For Sry replacement, we chose Sox9 (Sry-
related high-mobility–group box gene 9), a di-
rect target of SRY (2). Prior work showed that
transgenic overexpression of Sox9 driven by the
Wt1 promoter results in female-to-male sex re-
versal in XX mice (3). We placed the Wt1-Sox9
transgene in the context of a single X chromo-
some carrying the Eif2s3y transgene (fig. S1A)
(4) and found that it generated males (XEOSox9).
In these males, the Y-chromosome gene contribu-
tion is limited to Eif2s3y (table S1).
XOSry males, which carry an autosomally en-
coded Sry transgene, develop testes containing
spermatogonia that are unable to proliferate,
which results in seminiferous tubules appearing
empty when compared with those from males
with an intact Y chromosome (XY) (Fig. 1, A and
B). This defect can be overcome by transgenic
Eif2s3y addition to the X chromosome (XEOSry)
(table S1 and fig. S4A) (1, 5). To replace Eif2s3y,
we transgenically overexpressed its X chromosome–
encoded homolog, Eif2s3x (fig. S2). We then placed
the Eif2s3x transgene in the context of XOSry
(fig. S1B and supplementary text). The result-
ing XOSry,Eif2s3x males (carrying autosomally
1
Institute for Biogenesis Research, John A. Burns School
of Medicine, University of Hawaii, 1960 East-West Road,
Honolulu, HI 96822, USA. 2Aix-Marseille Université,
INSERM, GMGF UMR_S 910, 13385 Marseille, France.
*These authors contributed equally to this work.
†Corresponding author. E-mail: mward@hawaii.edu
encoded Sry and Eif2s3x transgenes) had the
Y-chromosome contribution limited to Sry (table S1).
XEOSox9 and XOSry,Eif2s3x males had small
testes (fig. S3), but spermatogenesis was ini-
tiated and progressed through meiosis and ar-
rested at the round spermatid stage (fig. S4, B
and C). Spermatogonia/Sertoli ratios in XEOSox9
and XOSry,Eif2s3x and spermatid/Sertoli ratio
in XEOSox9 were comparable to XEOSry but
lower than those in XY (Fig. 1, D and E). Round
spermatids in XOSry,Eif2s3x were dramatical-
ly depleted; XEOSry and XY had 10 and 88 times
as many, respectively (Fig. 1E and table S2). The
spermatids from both XEOSox9 and XOSry,Eif2s3x
males were functional in assisted fertilization,
and live offspring were obtained after embryo
transfer (Table 1).
We next tested whether spermatogenesis can
take place in males with a complete absence of
Y-chromosome genes. We used the same trans-
genes that were successful in single–Y gene sub-
stitutions (Wt1-Sox9 and Eif2s3x Tg1) to generate
mice transgenic for Sox9 and Eif2s3x in the XO
context (XOSox9,Eif2s3x) (fig. S1C and table S1)
The majority (35 out of 48) of XOSox9,Eif2s3x
males had testicular defects and essentially no
germ cells (fig. S5 and supplementary text). In
the remaining males, spermatogonial prolifera-
tion arrest was overcome (Fig. 1C and fig. S5),
and spermatogenesis progression was compa-
rable to that of XOSry,Eif2s3x (Fig. 1, D and E,
and table S2). Using assisted reproduction [round
spermatid injection (ROSI)], we injected oocytes
with spermatids from 13 males and obtained
zygotes with two well-developed pronuclei and
normal two-cell embryos (fig. S6, A to C, and
movie S1). Embryos from 11 males were used for
transfer. Ten resulted in pregnancy, and nine yielded
offspring (Table 1). Among the males that yielded
progeny, there were F1, F2, and F3 generation
XOSox9,Eif 2s3x ROSI males (fig. S6D). ROSI
offspring from males with one or no Y-chromosome

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 RE S EAR CH | R E P O R T S

Fig. 1. Testis histology analysis.Tubules of periodic

acid–Schiff–hematoxylin–stained sections of testis
from XY (A), XOSry (B), and XOSox9,Eif2s3x (C) males.
XY have normal spermatogenesis with expected
germ cell types present, including step 16 sperma-
tids [inset (A)]. XOSry have spermatogonial prolif-
eration arrest, resulting in tubules lacking germ
cells except for occasional normal [inset (B)] and
abnormal (*) spermatogonia. XOSox9,Eif2s3x have
meiotic and postmeiotic arrests that occasionally
allow formation of round spermatids [insets (C)],
arresting at step 7. Scale bar, 50 mm; insets, ×3 mag-
nification. (D and E) Quantitative analysis of sper-
matogenesis progression. Bars are averages ± SEM,
with n under the x axis. Statistical significance (t test):
(D) bars marked with a are different from all others
(P < 0.05), **P < 0.01; (E) bars with different letters
are significantly different (P < 0.05).

Downloaded from http://science.sciencemag.org/ on March 18, 2021

Table 1. The results of ROSI with spermatids from males with a single or no Y-chromosome genes. Column heads 2 to 4: Males with round spermatids
identifiable in live testicular cell suspension out of males examined. Males yielding progeny out of the number of males that yielded embryos used for embryo
transfer and induced pregnancy. Live offspring as a percentage of embryos transferred. Statistical significance (Fisher’s exact test, P < 0.05): aDifferent from
all others.

Males with round spermatids Live offspring

Male genotype Y gene contribution Males yielding progeny
(%) (no.) (%) (no.)
E
X OSox9 Eif2s3y 80 (8/10) 7/8 15.7 (18/115)
............................................................................................................................................................................................................................................................................................................................................
XOSry,Eif2s3x Sry 55 (17/31) 16/17 22.5 (57/253)
............................................................................................................................................................................................................................................................................................................................................
XOSox9,Eif2s3x None 27 (13/48)a 9/10 20.7 (46/222)
............................................................................................................................................................................................................................................................................................................................................
XY control Intact Y 100 (6/6) 6/6 25.0 (22/88)
............................................................................................................................................................................................................................................................................................................................................

genes were all normal and healthy (figs. S7 to S9 able to take over the Sry function in sex deter- matogenesis, but each homolog has evolved a
and supplementary text). mination. Manipulation of expression of other distinct expression level. Eif2s3y transcript amounts
The quantification of Eif 2s3x/y transcripts genes can lead to sex-fate change [reviewed in are ~5 to 7 times those in premeiotic and meiotic
in males transgenic for Eif 2s3y or Eif 2s3x re- (6–8)]. A surrogate sex-determination mecha- cells (11), which explains why the addition of one
vealed a correlation between spermatogenesis nism can also be activated without human input, Eif 2s3x transgene copy could not replace the
progression and Eif 2s3x/y expression level (Fig. as shown by two rodent species that lost the Y function of endogenous Eif2s3y in driving sper-
2, figs. S10 to S12, and supplementary text). All chromosome and Sry (9, 10). matogenesis through meiosis. Our finding that
transgenic males had their respective transgene Eif 2s3y and Eif 2s3x represent a typical, for- a single Eif2s3x transgene copy was sufficient to
transcript levels elevated when compared with merly autosomal, single-copy, X-Y homologous substitute for Eif2s3y in overcoming spermato-
XY (Fig. 2, A and B). Compared with Eif 2s3x gene pair and were hypothesized to have inter- gonial proliferation arrest suggests that the strong
transgenic males, Eif2s3y transgenic males showed changeable function (1, 5). Our data support this Eif2s3y expression in spermatogonia is required
higher Eif 2s3x/y transcript levels (Fig. 2C) and hypothesis: A single additional copy of Eif2s3x for the subsequent meiotic stages but not for
increased incidence of round spermatids (Fig. 1E). can functionally replace Eif 2s3y in spermato- mitotic proliferation. Our observations contradict
When spermatogenesis and Eif2s3x expression genesis initiation. For progression through meio- the accepted dogma of X-Y gene pairs evolving
were examined in XOSry,Eif 2s3x males with sis, however, at least four Eif2s3x transgene copies by decay on the Y chromosome and compensa-
varying numbers of Eif 2s3x transgene copies, are necessary, and the number of global Eif2s3x/y tion on the X chromosome (12), because here,
one (Tg2 and Tg6) and four (Tg1), no differences transcripts must reach a certain threshold. Our it is a beneficial overexpression of the Y gene
in spermatogonia/Sertoli cell ratio were observed, data also suggest that Eif 2s3x/y may play roles Eif2s3y and not its X homolog that appears to
but round spermatids were found only in Tg1 in gonad formation. We observed severe abnor- have evolved to meet the needs of spermato-
males, in which Eif 2s3x transcript levels are 2.4 malities of mature testes, indicative of impaired genesis. This might be the result of a selective
to 2.9 times those in Tg2 and Tg6 males (Fig. 2, gonadal development, in XOSox9,Eif2s3x but advantage during oogenesis for reduced Eif2s3x
D to F, and fig. S4, D to G). not XEOSox9 males. Because the global Eif2s3x/y levels or a selection for male germ cell beneficial
We have shown that a male mouse without expression is lower in the former, this suggests effects on the Y chromosome.
any Y-chromosome genes but with transgenically that a critical level of Eif 2s3x/y may be required It is generally believed that widely expressed
activated Sox9 and Eif2s3x can generate haploid for efficient testis differentiation. genes on the human Y chromosome with X homo-
gametes and father offspring with the help of Our data support a model where Eif 2s3y and logs that escape X inactivation are dosage-sensitive
assisted fertilization. Sox9 is not unique in being Eif 2s3x are functionally interchangeable in sper- (13). Dosage sensitivity explains why genes are

SCIENCE sciencemag.org 29 JANUARY 2016 • VOL 351 ISSUE 6272 515

R ES E A RC H | R E PO R TS
Fig. 2. Relation between Eif2s3x/y expression and spermatogenesis progression. (A to C) Transcript levels of endogenous and transgenic sper-
matogonial proliferation factors quantified by real-time polymerase chain reaction with Actb as a loading control and XY serving as reference control.
(D to F) Analysis of spermatogenesis progression (D and E) and Eif2s3x expression (F) in XOSry,Eif2s3x males with 4 (Tg1) and 1 (Tg2 and Tg6) Eif2s3x
transgene copies. Means ± SEM, with n under the x axis; bars with different letters are statistically different (t test, P < 0.05).
conserved on the Y chromosome: A certain com-
bined X-Y dose is critical at certain stages in cer-
tain tissues, and the X-gene dose cannot simply
be increased globally to compensate for the loss
of the Y gene because this would have detrimen-
tal effects in females. To lose the Y gene safely,
the X gene, or the genome, or the developmental
systems must adapt. Possible strategies might
include incremental increases in X-gene dosage,
relaxing constraints on dose-sensing, or retro-
genes (14). The mouse Eif2s3x/y gene pair is not
sensitive to overexpression; substantial elevation
of Eif2s3y (5) or Eif2s3x in the XY context (this
study) has no obvious somatic effects, nor does it
affect spermatogenesis and fertility. These find-
ings suggest that dosage sensitivity may appear
mainly in association with underexpression and/
or may vary between different X-Y gene pairs.
Altogether, our analyses of the Eif2s3x/y gene
pair support their importance for spermatogen-
esis. It will now be imperative to explore the
mechanisms whereby Eif2s3x/y factors exert their
functions during testicular development and sper-
matogenesis. Our work also paves the way and
prompts future evaluations of other ancestral
X-Y gene pairs to clarify the dosage requirements
516 29 JANUARY 2016 • VOL 351 ISSUE 6272
for spermatogenesis and beyond. Finally, our dem-
onstration that offspring can be obtained from
males with no Y-chromosome genes shows that for
assisted reproduction in the mouse, the Y chromo-
some is no longer necessary. However, there is
extensive evidence from both phenotype charac-
terization (15–17) and genomic analyses (13, 14, 18)
unequivocally supporting the importance of
Y-chromosome genes for normal, unassisted fer-
tilization. So, although our data demonstrate that
it is possible to bypass the requirement for the
Y chromosome in male assisted reproduction,
the Y clearly remains the genetic determinant of
full natural masculinity.
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AC KNOWLED GME NTS
The work was supported by NIH HD072380 and Hawaii Community
Foundation 14ADVC-64546 grants to M.A.W. and INSERM core
funding to M.J.M.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/351/6272/514/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S12
Tables S1 to S4
References (19–50)
Movie S1
4 August 2015; accepted 14 December 2015
10.1126/science.aad1795
sciencemag.org SCIENCE
Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction
Yasuhiro Yamauchi, Jonathan M. Riel, Victor A. Ruthig, Egle A. Ortega, Michael J. Mitchell and Monika A. Ward

Science 351 (6272), 514-516.

DOI: 10.1126/science.aad1795

Replacing the Y chromosome

The mammalian Y chromosome encodes a specialized set of genes that are essential for male viability and
fertility. In particular, the sex-determining region Y (SRY) protein is necessary to initiate male sex determination.
However, Yamauchi et al. show that the functions of the entire Y chromosome can be replaced with only two genes. In
mice, two transgenes, Sox9 and Eif2s3x, compensated for the absence of all Y chromosome genes to allow successful

Downloaded from http://science.sciencemag.org/ on March 18, 2021

sperm formation.
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REFERENCES This article cites 49 articles, 8 of which you can access for free
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